Watermelon, Citrullus lanatus (Thunb.) Matsum. & Nakai (2n=2x=22), belongs to the botanical family Cucurbitaceae. It is an important specialty crop accounting for 7% of the world area devoted to vegetable crops.
The goal of vegetable breeding is to combine various desirable traits in a single variety/hybrid. Such desirable traits may include greater yield, resistance to insects or pests, tolerance to heat and drought, desired earliness, seedlessness, better agronomic quality, higher nutritional value, growth rate, fruit flesh quality and fruit properties.
Cultivated watermelon (Citrullus lanatus var. lanatus) is a member of the Cucurbitaceae family. The plant is a large and sprawling annual, grown for its fruit. The fruit flesh is commonly red. Diploid watermelon fruits contain black seeds, which are considered undesirable for certain uses. Triploid watermelon fruits, produced by pollinating triploid plants with diploid pollen of pollenizer plants, are seedless.
Many different watermelon cultivars have been produced, and watermelon breeding efforts have been underway in many parts of the world. Some breeding objectives include varying the color, texture and flavor of the fruit, and absence of seeds. Other objectives include disease or pest resistance, optimizing flesh thickness, yield, suitability to various climatic circumstances, solid content (% dry matter), and sugar content.
The market for fresh-cut watermelon has increased at a rate of 10-30% annually for the last decade and the fresh-cut product now accounts for 13% of total fresh cut sales (USA). Fresh-cut watermelon is marketed as halves, quarters and slices with rinds, or as rind-free chunks. Quality degradation has been associated with decreased acceptability of texture, color and sweetness, with shelf-life limited by water soaking, juice leakage, off-odor development and increased microbial growth and spoilage.
The advantage of fresh-cut watermelon displays is that consumers can visually assess the quality of the product. Overripe fruits may show signs of decay, while unripe fruits are not uniform in fruit flesh pigmentation. Juice leakage is also a parameter that is associated with decaying fruit freshness.
Cutting watermelon fruits is known to have an impact on fruit quality by softening or deterioration of the fruit flesh causing liquid leakage. Cutting watermelons therefor has a negative impact on the shelf life of the fresh cut watermelon fruit.
Breeding techniques take advantage of a plant's method of pollination. There are two general methods of pollination: a plant self-pollinates if pollen from one flower is transferred to the same or another flower of the same plant or plant variety. A plant cross-pollinates if pollen comes to it from a flower of a different plant variety.
Plants that have been self-pollinated and selected for type over many generations become homozygous at almost all gene loci and produce a uniform population of true breeding progeny, a homozygous plant. A cross between two such homozygous plants of different varieties produces a uniform population of hybrid plants that are heterozygous for many gene loci. Conversely, a cross of two plants each heterozygous at a number of loci produces a population of hybrid plants that differ genetically and are not uniform. The resulting non-uniformity makes performance unpredictable.
Watermelon fruit firmness is described in EP2443919 (Seminis Vegetable Seeds). EP2443919 discloses that there are no industry standards to describe the firmness of the edible portions of watermelon fruits. This has led to a wide range of descriptors in use, from “firm” and “crisp” (Erma Zaden catalog descriptors for varieties Gil 104 and Erma 12) to “very firm flesh” (Zhang et al. in USPTO application numbers 20040060085 and 20030217394 and Seminis watermelon catalog for the variety Cooperstown). Seminis has described cultivars Fenway, Royal Star and Sentinel as having “excellent crispness,” “firm flesh” and “crisp juicy flesh,” respectively. In addition, Rogers Seed Company advertises the Tri-X Brand 626 as “exceptionally firm” and the Tri-X Brand 313 as having “firm texture” and “crispness.”
Despite the difference in language used in advertisings, EP2443919 discloses that quantitative measurements show that typical commercial germplasm has substantially lower flesh firmness than the watermelon fruit of EP2443919. EP2443919 further discloses that measurements of the prior art can be confusing, commercial watermelon lines produced prior to EP2443919 have fruit firmness that is well below 3 lbf and that the fruit of such commercial watermelon lines, once cut, undergo significant liquid leakage.
EP2443919 (also published as WO2006 014463, EP1765059 and US2006/005284) however, does not disclose the genetics causing the firmness of the watermelon fruit. EP2443919 suggests that firm flesh variation is the result of polygenic inheritance caused by several quantitative trait loci (QTLs). Variation in the phenotype of a quantitative trait is the result of the allelic composition at the QTLs and an environmental effect. Various potential causes for the variation are disclosed: (1) the fruit firmness trait may be controlled by several to many QTLs; (2) the fruit firmness trait may be caused by one or a few genes, but have a low heritability; and (3) the trait may be both polygenic and have low heritability. The QTLs of EP2443919 originate from a wild South African accession, PI296341, of C. lanatus var. citroides, which has small white fruits.
US2013/055466 (also published as WO2013033611, EP2750495) discloses a watermelon plant comprising at least one ultra-firm watermelon flesh phenotype wherein the locus is in a genomic region flanked by loci NW0251464 and NW0250266. According to FIG. 3A this locus is located on chromosome 9, though comparison of the marker loci NW0251464 and NW0250266 with the watermelon genome, suggests the locus is located on chromosome 6. The QTL also is an introgression from the same wild accession, PI296341, as mentioned above.
It is evident that the utility of an invention (in this case a specific trait in plant breeding such as fruit firmness) is higher when the trait can easily be transferred from one plant to another within the same species. A lower number of QTLs or one major QTL responsible for a specific trait is therefore preferred by plant breeders. Especially preferred are traits (e.g. plant phenotype characteristics) that are caused by a single gene. Traits caused by a mutation in a single endogenous gene of a cultivated plant (i.e. in cultivated germplasm of C. lanatus var. lanatus) has great advantages over QTLs identified in wild germplasm, such as PI296341 (C. lanatus var. citroides), because introgressions of QTLs from wild germplasm are very laborious and normally undesirable genetic regions are co-transferred into the cultivated germplasm together with the QTLs.
There is thus a need for watermelon plants that produce watermelon fruits that have an high texture (firm) fruit flesh, especially caused by one or more mutations in a single endogenous C. lanatus var. lanatus gene, and not by introgressions from wild germplasm, such as C. lanatus var. citroides. Thus, a mutation in an endogenous gene is herein referred to as a mutation in a gene of cultivated watermelon.
It is an object of the invention to provide a genetic cause for the high texture phenotype in cultivated watermelon (C. lanatus var. lanatus). The gene responsible for high texture (high texture gene) in cultivated watermelon was named C1MBP 17_2, for (Citrullus lanatus Mads Box Protein 17_2). It is also an object to provide cultivated watermelon plants, which produce fruits having an high texture fruit phenotype, conferred by one or more mutations in the endogenous (C. lanatus var. lanatus) high texture gene C1MBP 17_2, whereby said mutations lead to a reduced expression of the gene (and reduced levels of the encoded protein), or a reduced function or activity of the encoded protein. It is a further object to develop one or more markers that can be used in the selection of plants comprising one or more mutations in the high texture gene, resulting in a high texture phenotype. Also methods for either generating or for identifying plants or plant parts comprising alleles of the gene conferring the high texture fruit phenotype are provided.